Fumaric acid fermentation process



United States Patent 2,861,922 Patented Nov. 25, 1958 fifice 2,861,922 FUMAnrc ACID rnm isNrArIoN PROCESS Hyman R. iLubowitz and Earl G. La Roe, Cincinnati, Ohio, assignors to National Distillers and Chemical Corporation, New York, N. Y., a corporation of Virginia No Drawing. Application December 21, 1955 Serial No. 554,413

ll) Claims. (Cl. 19536) The present invention relates to an improved process for the production of fumaric acid from various carbohydrates and carbohydrate-containing substances by means of fumaric acid-producing fungi.

It is known that fumaric acid can be produced from a variety of carbohydrate substances by means of certain fungi; for example, in Patent Nos. 2,327,191 and 2,326,896, processes are disclosed whereby fermentable carbohydrate solutions containing suitable nutrient materials are fermented by fumaric acid-producing fungi to produce fumaric acid or, when carried out in the presence of a suitable neutralizing agent, to produce salts of fumaric acid. As disclosed therein, fungi belonging to the genus Rhizopus, including Rhizopus nz'gricans, are useful for such a purpose as are certain other organisms belonging to the order of Mucorales such as species of Mucor, Cunninghamella and Circinella. Still other fungi contemplated for such usage include some fumaric acidproducing Aspergilli and other strains belonging to the genus Rhizopus and including Rhizopus oryzae and Rhizopus arrhizas. Thus, it is known that numerous fungi possess the capacity to form fumaric acid by fermentation of carbohydrate solutions whereby, as used herein, the term fumaric acid-producing fungi is intended to include fumaric acid-producing organisms as set forth hereinbefore as Well as other organisms which, though not specifically referred to herein, also possess the capacity to produce fumaric acid by fermentation of a variety of fermentable carbohydrate-containing substances including sugars, starches and their derivatives; more specifically, glucose, fructose, sucrose, invert sugar, maltose, invert high test molasses, syrups, and various starches, grains, malted grains, cereal products and other materials containing any of the foregoing substances.

In carrying out the process for fermentation of fermentable carbohydrates by means of fumaric acid-producing fungi, the fermentation (i. e., the fumaric acid production stage) is carried out in presence of suitable nutrient materials. For example, and for use as nutrients in the production stage, salts of potassium, salts of magnesium and nitrogen-containing substances may suitably be employed in various combinations and concentrations with typical nutrient composition being composed of potassium acid phosphate, magnesium sulfate and ammonium sulfate. With respect to the nitrogen-containing nutrient, ammonium sulfate is particularly suitable but other nitrogen-containing substances may be employed and illustrative of which are urea, ammonium nitrate, ammonium chloride, aqueous ammonia, and the like.

in such processes for production of fumaric acid, a dcsiderata is the obtainment of fumaric acid in increased yields particulariy when carrying out the fermentation under standardized specified conditions, and, in certain instances, even relatively small increases in yield are of considerable importance in rendering a particular process more feasible economically or conversion of an otherwise uneconomical process to one that is commercially practicable. The primary object of this invention is the proviweight of nickel io-n per sion of a process for production of fumaric acid by means of fumaric acid-producing fungi whereby substantially increased yields of fumaric acid are obtained.

The process of the present invention comprises the production of fumaric acid by fermentation of a fermentable carbohydrate by means of fumaric acid-producing fungi in the presence of a nickel-containing substance. It has been found, as is more clearly described hereinafter, that by carrying out the fermentation in presence of a nickelcontaining substance the yield of fumaric acid is substantially improved over the yields of fumaric acid obtained by carrying out the fermentation in similar or identical manner but in the absence of nickel.

The fermentation process embodied herein is generally carried out in the presence of a nitrogen-containing nutrient. For such a purpose, the total amount of nitrogen normally required as a nutrient in fumaric acid-producing processes may be varied depending on factors such as the particular carbohydrate or carbohydrate-containing substances subjected to the fermentation treatment, the presence of other metallic ions in the fermentation medium, and the like, but usually the fermentation may suitably be carried out by use of a nitrogen-containing nutrient in a ratio of one part of nitrogen to from about 25- to about 300 parts of carbohydrate (as carbon). Thus, as applied to fermentation of carbohydrate materials by means of furnaric acid-producing fungi for production. of fumaric acid under standardized conditions including temperature and ingredients of the fermentation medium, the amount of total nitrogen required as a nutrient for carrying out the fermentation to optimum yields of fumaric acid can be determined by carrying out a series of such fermentations with addition, to the production medium, of varying amounts of nitrogen at the start of the fermentation and determination of the amount of total nitrogen that produces optimum or substantially optimum yields of fumaric acid under the conditions employed.

As aforesaid, and in accordance with this invention, it has been found, that, in carrying out a fermentation process as aforedescribed under otherwise identical or similar conditions, substantially improved yields of fumaric acid can be obtained if the fermentation is carried out in the presence of a source of nickel ions. More specifically, and in preferred aspects of the invention, the fermentation process as embodied herein is carried out by having present, from about 10 to about 300 parts. by million parts of the fermentation medium and more preferably, the fermentation process embodied herein is carried out by having present in the fermentation medium from about 40 to about parts of nickel ion per million parts of the fermentation medium.

With reference to the aforedescribed process, the source of nickel ions in the fermentation medium may be provided by incorporating a salt of nickel in amounts that provide an improved yield of fumaric acid as compared to carrying out of the fermentation under otherwise identical or similar conditions, but in the absence of added nickel ions. For such a purpose, salts of nickel such as nickel chloride, nickel sulfate, nickel carbonate, and the like may be employed. Other sources ofnickel ions contemplated for use herein include nickel organic compounds such as nickel oleate, nickel acetate, and the like. Although, in general aspect, the amount of nickel employed in the fermentation medium is such that a substantial increase in fumaric acid yield is provided, the amount of nickel usual.- ly employed with suitable results is Within the aforestated ranges in parts of nickel ion per million parts of the fermentation medium.

In order to illustrate practice of the invention, specific embodiments thereof are set forth hereinafter in which the fermentable carbohydrate employed was an inverted high test molasses, the fumaric acid-producing fungus was a culture of the genus Rhizopus, the nitrogen-containing nutrient was ammonium sulfate and the source of nickel was nickel chloride (NiCl bH O). Although such specific materials have been utilized in the described embodiments, it should be understood that their usage is for the purpose of illustrating but not limiting the invention thereto.

Preparation of fumaric acid-producing fungus.-A culture of Rhizopus species (strain of Rhizopus oryzae) was grown for 24 hours at 2830 C. with shaking (i. e., submerged culture) in an aqueous medium of the following composition (grams/ 100 ml.):

Invert molasses 2.0 Calcium carbonate 0.8 Ammonium sulfate 0.2 Magnesium sulfate heptahydrate 0.05 Potassium acid phosphate 0.05 Zinc sulfate heptahydrate .0004- Ferric sulfate .0004

Example 1 Preparation of fermentation medium.A fermentation medium was prepared by inoculating, with 10.5 mls. of the culture of Rhizopus species obtained as aforedescribed, a production substrate prepared as follows: 15.0 grams of invert sugar (produced by enzymatic or acid inversion of high test molasses), 9.75 grams of calcium carbonate, 0.1125 gram of ammonium sulfate (plus 0.075 gram on second and fourth days of fermentation), 0.0375 gram of magnesium sulfate heptahydrate, 0.075 gram of potassium acid phosphate, 6.0 mgs. zinc sulfate lieptahydrate, l2.0 mgs. of ferric sulfate hydrate, 15 mgs. manganese sulfate and sufficient water to make 140 mls. were mixed in a 500 m1. Erlenmeyer flask and the contents of the flask were sterilized. The fermentation medium, prepared as aforedescribed, was fermented with agitation at 2830 C. for periods of time set forth in the following tabulation. The following tabulation also sets forth the yield of fumaric acid obtained by carrying out the fermentation in the absence of an added source of nickel ions as compared to the results obtained by carrying out the fermentation under identical conditions except that nickel chloride in the amount shown was added to the fermentation medium. The addition of the nitrogen source in three increments may be substituted for by addition of the total nitrogen requirement at the beginning of fermentation or by other incremental addition schedules.

Nickel Chloride Added, Fermen- Yield of Percent P. p. m. of tation Fumarlc Run No. (Based on Ni++ time, Ac

weight of days Percent 1 Fermentation medium) 1 Based on amount of sugar present at beginning of fermentation. I No zinc sulfate added to medium. 8 24 mgs. ferric sulfate hydrate per flask.

As is apparent from the data in the foregoing tabulation, in each instance wherein the nickel-containing compound was added to the fermentation medium, the yield of fumaric acid was increased as compared to the yield of fumaric acid obtained by carrying out the fermentation under identical conditions but without addition of the source of nickel ions.

In practice of the invention, the fermentation process is carried out at temperatures that usually may range from about 28 to about 34 C. with a temperature of about 28 C. being particularly suitable. However, the temperature utilized may be varied to those somewhat lower or higher than the aforesaid range depending on factors such as the specific fungus employed in the production medium as well as on the other ingredients and concentrations thereof present in the particular fermentation medium employed. Moreover, and in accordance with conventional practice, the fermentation process as embodied herein may be carried out in the presence of a suitable neutralization agent whereby the fumaric acid that forms during the fermentation is neutralized so as to provide for more rapid conversion of the fermentable substances to fumaric acid. For such usage, neutralizing agents such as calcium carbonate, sodium or potassium hydroxides, etc. are suitable.

As is apparent from the foregoing description, the invention embodied herein is directed to an improvement in the fumaric acid-producing stage, as distinguished from the fungus growth stage, in fumaric acid-producing processes by fermentation of fermentable carbohydrates, such as sugar solutions, by means of fumaric acid-producing fungi. For example, the fungus utilized in practice of this invention may be prepared by growing the culture in presence of certain carbohydrate materials such as invert molasses, black strap molasses, etc. that contain growth stimulating substances or by growing the culture in sugar solution, such as glucose, sugar syrup, etc. to which growth stimulants are added, such for example, as disclosed in Patent No. 2,326,986. Moreover, and as to the preparation of the organisms for inoculation of the fermentation medium, the inoculum may be produced in a single growth stage or a plurality of growth stages. For example, the organisms may be grown in a single stage as is illustrated by the procedure used in preparation of the Rhizopus species described in the embodiment herein or the organisms may be grown in a plurality of stages. In illustration of the latter method for preparation of an inoculum, a sporulated culture of a suitable fumaric acid-producing strain of Rhizopus on a liquid or solid growth medium is used to seed a liquid growth substrate of suitable composition. After incubation under conditions of agitation and at a suitable temperature, and with provisions for aeration of the substrate, 'the submerged culture of the fungus is developed into a dispersed, filamentous mass whereby the culture is capable of producing fumaric acid when inoculated to a production substrate. However, a number of subcultures or serial trans fers of the primary submerged inoculum may be prepared in media of similar or identical composition. For example, a submerged fungus culture grown in aforedescribed manner may be inoculated into a liquid growth substrate. After sufficient incubation at a temperature between 2834 C. with provisions for aeration and agitation the resultant filamentous mass of fungus is again suitable for use in production of fumaric acid when inoculated to a production substrate. A third or fourth stage inoculum may then be prepared in a similar manner by increasing, decreasing or holding constant the volume of substrate. By increasing the volume of substrate stepwise and employing the previous stage of inoculum so that the inoculation constitutes between 0.5 and 25 percent of the total stage volume, such a procedure may be used to build up a suflicient amount of submerged fungus mycelium for large scale fermentation.

While there are above disclosed but a limited number of embodiments of the invention herein presented, it is possible to produce still other embodiments without departing from the inventive concept herein disclosed, and it is desired therefore that only such limitations be imposed on the appended claims as are stated therein.

What is claimed is:

1. In a process for production of fumaric acid by fermentation of a carbohydrate-containing substance by means of fumaric acid-producing fungi of the genus Rhizopus, the improvement which comprises carrying out said fermentation in the presence of a source of nickel ions in an amount of from about to about 300 parts of nickel ions per million parts of the fermentation medium.

2. A process, as defined in claim 1, wherein the source nickel ions is a nickel salt of an inorganic acid.

3. A process, as defined in claim 1, wherein the fermentation is carried out in presence of nickel ions in an amount of from about 40 to about 180 parts per million parts of the fermentation medium.

4. In a process for production of fumaric acid by fermentation of a sugar solution by means of a fumaric acidproducing fungus of thestrain of Rhizopus oryzae, the improvement which comprises carrying out said fermentation in the presence of nickel ions in an amount of from about 10 to about 300 parts per million parts of the fermentation medium.

5. In a submerged culture process for production of fumaric acid by fermentation of a carbohydrate-containing substance by means of fumaric acid-producing fungus of the genus Rhizopus in presence of a nitrogen-containing nutrient, the improvement which comprises carrying out said fermentation in presence of a source of nickel ions in an amount of from about 10 to about 300 parts of nickel ions per million parts of the fermentation me dium.

6. A process, as defined in claim 5, wherein the source of nickel ions is a nickel salt of an inorganic acid.

7. A process, as defined in claim 5, wherein the nickel salt is nickel chloride.

8. A process, as defined in claim 5, wherein the fumaric acid-producing fungus is an organism of the strain of Rhizopus oryzae.

9. In a process for production of fumaric acid by fermentation of a sugar solution by means of a fumaric acidproducing organism of the genus Rhizopus in presence of ammonium sulfate, the improvement which comprises carrying out said fermentation in presence of nickel ions in an amount of from about 10 to about 300 parts per million parts of the fermentation medium.

10. A process, as defined in claim 9, wherein the fermentation is carried out in presence of nickel chloride in an amount to provide from about 10 to about parts of nickel per million parts of fermentation medium.

References Cited in the file of this patent UNITED STATES PATENTS I Waksman Aug. 17, 1943 Kane et al Aug. 17, 1943 OTHER REFERENCES No. 4, April 

1. IN A PROCESS FOR PRODUCTION OF FUMARIC ACID BY FERMENTATION OF A CARBOHYDRATE-CONTAINING SUBSTANCE BY MEANS OF FUMARIC ACID-PRODUCING FUNGI OF THE GENUS RHIZOPUS, THE IMPROVEMENT WHICH COMPRISES CARRYING OUT SAID FERMENTATION IN THE PRESENCE OF A SOURCE OF NICKEL IONS IN AN AMOUNT OF FROM ABOUT 10 TO ABOUT 300 PARTS OF NICKEL IONS PER MILLION PARTS OF THE FERMENTATION MEDIUM. 